Tissue sections are a type of biological sample that may be studied using histochemical techniques. Histochemical analysis of tissue samples often involves immunohistochemical staining to visualize features of the tissue for examination under a microscope.
Creating tissue sections may involve freezing the tissue or preserving the tissue with a fixative and supporting the tissue structure by infiltrating the tissue with paraffin wax. Paraffin has been used for many years as an embedding medium in the preparation of tissue specimens for sectioning in a microtome to produce specimen sections for histological studies. Such embedding processes generally include specimen fixation, dehydration, clearing, paraffin infiltration or impregnation, blocking or embedding in a block of paraffin, slicing the block and specimen into thin sections, mounting the sections on microscope slides, removing the paraffin (“dewaxing” or “deparaffinizing”), partially reversing specimen fixation (“antigen retrieval”), and staining the sections prior to microscopic analysis. The primary purpose of freezing or of the embedding medium is to permit the specimens to be sectioned and mounted in a natural state. A common fixative is a formaldehyde-based solution such as formalin that cross-links proteins in the tissue specimen by forming methylene bridges which increase structural stability and help the specimen retain its natural structure. Sectioned tissues preserved in this way are referred to as formalin fixed paraffin embedded (FFPE) tissue sections.
Dewaxing of tissue sections is typically performed at room temperature using a volatile solvent like xylene or less teratogenic substitutes that dissolve the wax directly. Alternatively, dewaxing can be accomplished using an aqueous detergent solution by heating the slide above the melting point of the wax (the melting point varies based on composition of the wax and other factors but is between about 30 and 100° C.), which allows the detergent solution to emulsify the wax over several minutes. Dewaxing allows further treatment of the tissue section using aqueous reagents.
The stains used to color the tissue often include antibodies selected to bind to specific portions of the tissue. However, the fixation process may mask antigenic sites. Fixation can alter protein biochemistry such that the epitope of interest is masked and can no longer bind to the primary antibody. Masking of the epitope can be caused by cross-linking of amino acids within the epitope, cross-linking unrelated peptides at or near an epitope, altering the conformation of an epitope, or altering the electrostatic charge of the antigen. “Antigen retrieval” (AR) refers to any technique in which the masking of an epitope is reversed and epitope-antibody binding is restored. AR at least partially reverses some of the effects of fixation and exposes antigenic sites, allowing antibodies and thus, the stain to bind. The need for AR depends on multiple variables, including but not limited to, the target antigen, the antibody used, the type of tissue, and the method and duration of fixation.
Heat may be employed in AR, where one technique is to immerse the tissue section in a basic or acidic AR solution held between 95-115° C. for a period of 30-45 minutes. The choice of basic or acidic AR solution depends on which solution yields better reactivity with the staining antibody to be used subsequently. Immunohistochemical stains are applied to the tissue sample after AR.
Conventional automated systems for tissue staining on microscope slides heat the dewaxing and AR solutions using electric heating elements mounted under each slide. However, a glass microscope slide is a poor conductor of heat, requiring the use of relatively powerful (30-40 W) heaters under the microscope slides. The power demands of the heaters limit the capacity of automated systems. Further, the location of the heaters under the slides subjects the heaters to frequent direct exposure to moisture and caustic solutions during the tissue specimen preprocessing and staining processes, leading to heater failure. Because relatively low volumes (˜1 ml) of the dewax and AR solutions are typically dispensed on each slide, these liquids are subject to rapid evaporation, resulting in use of slide covers to reduce evaporation. Slide covers add process complexity and material waste.